title: In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation.
abstract: Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. m-jewett@northwestern.edu
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
authors.forename: Brian R
authors.initials: BR
authors.lastname: Fritz
authors.forename: Laura E
authors.initials: LE
authors.lastname: Timmerman
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D002499:Centrifugation, Density Gradient; D002981:Clindamycin / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering; D009154:Mutation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012338:RNA, Ribosomal, 23S / Q000235:genetics* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D011500:Protein Synthesis Inhibitors; D012338:RNA, Ribosomal, 23S; D012269:Ribosomal Proteins; D002981:Clindamycin